The medium was replaced almost every other day. The fast green fluorescent calcium indicator GCaMP6f open up reading body [2] was placed directly under the control of the CAG promoter, using a puromycin resistance gene and cloned into an AAVS1-concentrating on vector [12]. with myasthenia gravis, and created six recombinant antibodies. All AChR-specific antibodies were hypermutated, including isotypes IgG1, IgG3, and IgG4, and acknowledged different subunits of the AChR. Despite obvious binding, none of the individual antibodies showed significant antagonism of the AChR measured in an in vitro neuromuscular synapse model, or AChR-dependent match activation, and they did not induce myasthenic indicators in vivo. However, Mouse monoclonal to cTnI combinations of antibodies induced strong match activation in vitro, and severe weakness in a passive transfer myasthenia gravis rat model, associated with NMJ destruction and match activation BH3I-1 in muscle mass. The strongest match activation was mediated by combinations of antibodies targeting disparate subunits of the AChR, and such combinations also induced the formation of large clusters of AChR on the surface of live cells in vitro. We propose that synergy between antibodies of different epitope specificities is usually a fundamental feature of this disease, and possibly a general feature of complement-mediated autoimmune diseases. The importance of synergistic conversation between antibodies targeting different subunits of the receptor can explain the well-known discrepancy between serum anti-AChR titers and clinical severity, and has implications for therapeutic strategies currently under investigation. == Supplementary Information == The online version contains supplementary material available at 10.1007/s00401-022-02493-6. Keywords:Myasthenia gravis, IgG4, Match, Clustering, Human induced pluripotent stem BH3I-1 cells, Live cell imaging == Introduction == Myasthenia gravis (MG) is usually a debilitating autoimmune disease associated with autoantibodies against components of the synapses between motor neurons and muscle tissue (neuromuscular junctions, NMJ), making it one of the few autoimmune diseases in which the nature of the autoantigen provides an explanation for the symptoms. Numerous proteins can be involved, but four out of five patients [9,44] have antibodies against subunits of the acetylcholine receptor (AChR). The receptor is usually a ligand-gated ion channel of four closely related subunits, alpha (), beta (), delta () and epsilon (), each a four-pass transmembrane protein. Each receptor is usually a pentamer created by BH3I-1 two , and one each of the other subunits [42]. There is inter-individual variance in the proportions of autoantibodies targeting the four subunits of the receptor [21,43]. Action potentials arriving along the motor nerve result in the release of acetylcholine, which diffuses across the synaptic cleft of the neuromuscular junction, binds to the AChR and induces the opening of the channel, leading to depolarization and contraction of the muscle mass. Neural control of skeletal muscle mass is usually therefore completely dependent on the AChR, but how autoantibodies disrupt this process is not obvious. Three mechanisms have been postulated, namely: direct blockade of the receptor, destruction of BH3I-1 the receptor-bearing membrane by antibody-driven match activation, and depletion of the receptors by antibody-mediated cross-linking and internalization [6,7,23,40]. Sera from patients with anti-AChR-associated myasthenia gravis show evidence of all three of these mechanisms, in varying proportions [5,28,41]. Important improvements have been made by studying whole sera or crude antibody preparations extracted from sera [17,43], but understanding the relationship between antibodies and pathomechanisms requires examining the properties of individual patient-derived antibodies. For example, the isolation of antibodies against the muscle-specific kinase (MuSK), which are found in a small subset of myasthenic patients, has enabled the elucidation of their epitope specificity, and their effects on AChR clustering and MuSK phosphorylation [15,40]. The isolation of autoantibodies against AChRs can be achieved by similar methods [25], but this approach requires that this antigen be prepared in a soluble form. In the case of AChR, this is complicated by the multi-membrane-pass, heteropentameric nature of the antigen. We therefore developed methods for isolating B cells specific for AChR from MG patients, using intact, membrane-expressed AChR as bait antigen, and examined the pathogenic potential of their anti-AChR antibodies in molecular mechanistic detail. == Materials and methods == == Patients and healthy donors == Peripheral blood samples were collected from 12 healthy controls, six female and six male participants with an average age of 42, and 17 patients with clinically confirmed myasthenia gravis showing AChR-autoantibody RIA measurements above 0.5 nmol/l, with 6 female and 12 male participants and an average age of 62 (Supplementary Table 1). Peripheral blood was drawn BH3I-1 into S-Monovette tubes made up of 1.6 mg EDTA per ml blood (01.1605.100) for isolation of PBMC, and into S-Monovette tubes with clot activator (01.1601.100, both from Sarstedt) for serum preparation. PBMC isolation and serum preparation were performed as previously explained [1,47]. PBMC were stored in liquid nitrogen until use, serum was stored at 20 C. The project was examined and authorized by the Ethikkommission Nordwest und Zentralschweiz. == Plasmids and cell lines == TE671 rhabdomyosarcoma cells were obtained from ATCC (LGC, Wesel, Germany) and cultured in.